Human source leading sequence, gene vector and gene expression strategy

ABSTRACT

The present invention deals with the leading sequence of human source gene, gene vector and gene expression strategies. The invention include the following: (1) Using DNA sequence without pivot physiological function-related gene in short arms of human group D, G chromosomes, or a DNA sequence sharing 50% or over 50% identity to the sequence select from human D, G group chromosomes as leading sequence for gene targeting; (2) Construct the gene vector containing the leading sequence above; (3) Nucleolus organizing region in D, G group chromosomes mentioned in the procedure( 1 ) is used as the target site, the gene of interest is integrated into the short arms where the gene expresses actively in D, G group chromosomes of human cells. The present invention provides a novel gene leading sequence by which the gene vector construction and gene expression strategies are obtained. The gene expression strategies can be used for human gene therapy and manufacturing protein.

SUMMARY OF THE INVENTION

[0001] The invention deals with gene leading sequence, by which the gene vector was constructed and the expression strategy of target gene introduced by the human source gene vector.

BACKGROUND OF THE INVENTION

[0002] Statistic data of Mendelian inheritance in man demonstrates, up to now, that 1660 single gene disorders have been identified and 989 diseases-related genes have been identified before Jun. 30, 2000. Most of the recessive genetic disorders may be treated by introducing normal gene into cells of patient. With the research advancement, initiation of gene therapy regarding dominant genetic disorders and somatic cell -related tumors has begun. In 1995, American scholar E. Marshall put forward in Science that the key point of gene therapy research was novel vector development and discovery, but the problem of gene therapy vector remain unsolved so far. The main reason is that the researchers don't step out of circle that vector was constructed by viral components. General speaking, commonly-used viral vectors have many defects going as follows: 1) instability: gene insertion efficiency is low, partial vector existing in the cell nucleus as a form of attached body couldn't inherit stably with cell division, so that the therapeutic gene can't express long and stable. 2) safety is not good: For example, mutation caused by random insertion may affect the normal gene function in integration sites, and even activate oncogene, which may result in other diseases and tumors respectively. Furthermore, viral vectors may generate wild-type recombinant virus with replication ability do great harmful to the human body. In recent years, it was reported that adenovirus vector caused patient death during the manipulation of gene therapy. 3) Immunogenecity: the background protein produced by viral genes and protein contaminated during purification could induce immunogenic-reaction and influence the gene expression.

[0003] In the middle of 1980's, gene targeting vector was developed based on homology recombinant principle to achieved the site-directed integration, which could avoid immunogenecity and random integration, but gene targeting vector used for site-directed reparation in gene therapy and defect gene replacement have to utilize specific fragments of the two sides of the gene as leading sequences, therefore its application is limited and the transfection efficiency is still low. In fact, it is usually proper for gene knock-out of embryonic stem and fertilized egg cells, but not suitable for site-directed integration of mature somatic cells (Galli-Taliadoros LA, Sedgwick JW, Wood SA, et al. J Immunol Meth 1995, 181:1-15;Hasty P. Rivera-Perez J, Chang C, et al. Mol Cell Biol 1991, 11(9):4509-4517). Leon. E. Rosenbery, former president of American Human Gene Society, Dean of medical school of Yale University, concluded that no case had showed certain clinical I effective among hundreds of gene therapy experiments (Rosenbery L E & Schechter A. N.Science, 2000;287:1751). So that, to seek and develop a novel stable heredity gene vector with no harm to human body remain a key question to be solved.

DETAILED DESCRIPTION OF THE INVENTION

[0004] In 1981, the applicant found two families carrying a rarely reported bi-satellite microchromosome (BM), but the phenotype is normal. The microchromosomes steadily inherit in two families with 2 and 3 generations and show no harm to human body. Through document investigation, we found that there were seven similar families in both Europe and USA. Up to now, total 17 families have been reported, but no one thought of this chromosome to be components of human source gene vector construction. The inventor put forward in 1991 a proposal that disruption of this chromosome components and constitution of human source gene vector. The project was initiated in 1994 and executed in 1995. The applicant firstly detected during investigation that these chromosomes originated from short arms of human D and G group chromosomes including chromosome 13, 14, 15, 21 and 22. The short arms of D, G group chromosome contain nucleolus organizing region and are rich in ribosome DNA. Preliminary biological research has revealed that polymorphism of different lengths (namely containing different concentrations of rDNA) were broadly founded in this region in population, and the genes here could be transcribed very actively during cell diving. Therefore, the applicant infer that if we can isolate a specific fragment from BM and use it as a leading sequence, a foreign gene can then be transferred site-directly into nucleolus organizing region, the expression of the gene should be effective, stable and unharmful. The resultant experiments have strongly proved that assumption.

[0005] It is of great signification to find BM and isolate specific DNA fragment showing homologous to the nucleolus organizing region at short arms of human D, G group chromosomes.

[0006] The applicant constructed BM-specific pUC19 library by micro-dissection, PCR and microcloning techniques at first, and then isolated single copy fragment by screening this library. The single copy fragment was proved to be from BM and short arms of D, G group chromosomes by Fluorescent In Situ Hybridization (FISH). The single copy fragment was further used as probe to screen PAC genome library and gained a DNA fragment of 120 kb (BMSF) (SEQ No.1), which was also confirmed to originate from short arms of human D, G group chromosomes (FIG. 1). Sequence analysis indicated no physiological function related genes were found in this BMSF, suggesting the target site is safe.

[0007] The applicant realizes it is identical with invention aim to isolate a DNA sequence without pivot physiological function-related gene from short arms of human D, G group chromosomes or a sequence sharing 50% or great than 50% similarity to short arms in human D, G group chromosomes as gene leading sequence. For example, in example 1, a sequence was selected from the SEQ No. 1 to construct human source gene vector, this sequence comes from short arms of human D, G group chromosomes.

[0008] The 1.20 kb DNA fragment above can be used as gene leading sequence, and a smaller fragment with specificity selected from the 120 kb DNA fragment can also be selected. in the applied case, we select a 3.8 kb fragment from nucleotide 75590 to 79448 in the SEQ No. 1 as gene leading sequence (GLS). According to the requirements of gene vector construction, positive and negative screening genes should be added too.

[0009] More concretely, to construct a human gene therapy vector, the 3.8 kb fragment from nucleotide 75590 to 79448 shown in the SEQ No. 1 was inserted into pGEM-TK vector which contained a negative screen gene TK, positive screen gene Neo was inserted into site 1500 of GLS which divided GLS into two arms of 1.5 kb and 2.3 Kb, the gene vector were then constructed. The bacterial strain containing gene vector was reserved in China Typical Culture Collection Center on Sep. 29, 2000 (Wuhan University, Wuhan 430072, China). The reservation number is CCTCCM200030. The administrator designated the reserve classification nomenclature which is Escherichia coli JM109/JH-4/pNS2. The FIG. 2 shows the vector, and the sequence is given in the SEQ No. 2.

[0010] This present invention provides specific target site coining from SEQ No.1 which denotes DNA leading sequence originated from short arms of D, G group chromosome. No doubt, the gene vector constructed by fragment above can transfer gene of interest into specific target sites which are of no pivot physiological function-related genes, targeting is safe.

[0011] Based on invention technologies strategy, the applicant further put forward following procedures of gene expression strategies.

[0012] (1) Gene vector is constructed by DNA sequence with no vital physiological function -related genes in short arms of human being Group D,G chromosomes or by using a leading sequence which is 50% or great than 50% homologous to DNA sequence above;

[0013] (2) Clone gene of interest into gene vector above;

[0014] (3) Transfer gene of interest into target sites of nucleolus organizer region of group D,G chromosomes of host cells;

[0015] (4) Gene of interest expresses in vivo and in vitro.

[0016] All procedures including gene vector construction, recombination of gene of interest onto the vector, transfer of gene of interest into host cells and expression of gene of interest can be conducted by conventional technologies.

[0017] The leading sequence of gene of interest can be selected from DNA sequence of SEQ No. 1. Moreover, the leading sequence of the gene of interest is at the positions from 75590 to 79448 in SEQ No. 1.

[0018] The gene leading sequence above can be used to construct a gene vector containing positive and negative screening genes.

[0019] The example 2 and 3 of the present invention explained the procedures above:

[0020] {circle over (1)}better specific DNA fragment of 3.8 kb selected from BMSF of 120 Kb is subcloned into pGEM-TK plasmid vector, using TK as negative screen gene, meanwhile inserting positive screening gene Neo was inserted into 3.8 kb DNA fragment to construct gene Vector.{circle over (2)}gene of interest digested by enzyme is ligated into exogenous gene cloning site on one side of gene Neo .The polymerase chain reaction certified the existance of all parts and orientation. {circle over (3)}Select single enzyme digestion site in pGEM vector and linearized gene vector, transfer gene vector into target cells by means of electroporation or liposome transfection.{circle over (4)}Screen transformed target cells using G418 and GCV to obtain site-directed integrating positive cells clones.{circle over (5)}Detect the expression of gene of interest from positive cell clones as to reach site-directed integration into short arms of D, G group chromosomes and expression stably .

[0021] The positive cells expressing gene of interest screened are embedded hypodermatically or injected intravenously into body or the gene of interest encapsulated by liposome is directly injected into body so that gene can expresses long and stably within body to correct the clinical symptom caused by defect gene.

[0022] The example of the present invention provides DNA sequence of gene vector as shown as SEQ No. 2, of which leading sequence is from 75590 to 79448 of SEQ No. 1. TK is negative screening gene, the positive screening gene Neo is inserted into site 1500 of GLS and GLS is divided into twvo arms 1.5 kb and 2.3 Kb, cloning site is at nucleotide 5910.The example of the present invention made public in vitro expression in HT1080 cells of genes that tissue-type plasminogen activator (TPA) gene used for treatment of blood occlusion disorders and coagulant factor IX (FIX)gene used for treatment haemophilia B The experiment showed the expression is both efficient and stable.

[0023] Therefore, gene expression strategies provided by the present invention are of great promising in practical application. The gene expression strategies are not only used for manufacturing medicinal protein but also give an effective way for gene therapy.

[0024] The inventor made use of a chromosome being no harmful for the human being body as an unique material and create a completely novel strategy. It is the first that the inventor firstly found that short ann nucleolus tissue region of human D, G group chromosomes is the best target site for human gene therapy and expression(10 sites in short arms of nucleolus tissue region of chromosome 13, 14, 15, 21, 22) and construct a novel gene vector capable of transferring the gene of interest site-directly into these target sites.

[0025] Compared with background technologies, the present invention has some striking advantages:

[0026] 1. Good stability: using the DNA sequence provided by the patent claims 1 or 2 are gene leading sequence to construct vector, the human source gene vector can transfer site-directly the gene of interest into short arms of D, G group chromosomes of human somatic cell and allow gene of interest inherit stably with chromosomes;

[0027] 2. Good safety: target site containing no vital physiological function-related gene demonstrates that target site is safe. Meanwhile FISH confirmed the vector could insert site-directly gene of interest into safe target site in cells (FIG. 3, 4), excluding insertion mutation at random integration and harm of recombinant wild -type virus. The expression of gene of interest in target sites is safe. Although the present invention didn't provide clinical demonstration of gene expression strategies, the practical examples in section example 4 found by applicant and foreign researchers confirm its safety from another angle.

[0028] 3. Efficient expression: First, gene vector provided by The present invention comes from leading sequence at short arms of human D, G group chromosomes short arms, so correspondingly there are 10 target sites in human cells, the insertion efficiency is 5-10 times higher at least than any other vectors; Second, leading sequence coming from short arms of D, G group chromosomes where gene transcripts actively, the gene of interest delivered into target sites can express highly efficiently.

[0029] 4. No immunogenecity: the vector come from human being, it has no immunogenecity to human being.

[0030]FIG. 1 is mapping of 120 kb DNA fragment cloned in PAC by FISH

[0031]FIG. 2 is a structure map of gene vector (whole length of gene vector is 11162 bp); of which pGEM-7 (8267-11162): vector replication elements and prokaryotic screening system; TK(1-2840): Eukaryotic cell negative screening gene which utilizes TK promoter and TK poly A signal; Neo (4342-5910): eukaryotic cell positive screening gene which utilizes sv40 promoter and sv40 poly A signal; GLS(2841-434 1, 5911-8267): leading sequence; Cloning site(5910): insert site of the gene of interest;

[0032]FIG. 3 is a FISH result showing mapping of exogenous tPA gene in positive clone cells, which demonstrate the vector could target tPA gene site-directly into short arms of D, G chromosomes;

[0033]FIG. 4 is a FISH result showing mapping of exogenous FIX gene in the positive clone cells, which demonstrate the vector could target FIX gene into short arms of D, G group chromosomes;

[0034]FIG. 5 shows the western blot of purified tPA, lane 1-4 are purified product of tPA, “-” denotes to negative control;

[0035]FIG. 6 shows the western blot of FIX positive cells, F3-F23 are six different cell strains, “-” denotes negative control.

[0036] The examples provided in the present invention is one of the way to realize the present invention, can't be considered to be a limitation to the present invention.

EXAMPLE ONE

[0037] The preparation of the gene leading sequence provided in the present invention:

[0038] 1. Acquirement of PAC clone containing gene leading sequence

[0039] 1.1 Construct BM -specific pUC19 library through micro-dissection, PCR and microcloning technologies (Deng H-X, Yoshiura K, Dirks RW, et al. Hurn Genet 1992, 89:13.)

[0040] 1.2 Acquirement and identification of BM-specific single copy DNA

[0041] (1) The preparation of colony matrix membrane: Draw squares 14×14 on the two pieces of nylon membranes, mark A, B, place the two membranes on the two plates containing solid LB, respectively, pick at random white clones and transfer the clones into squares of two same coordinates, total 14×12 clones, single copy DNA of 100 ng is added to the line 13 as positive control, no addition of DNA as negative control. The two plates are respectively placed in incubator at 37C for 10-12 hr, membrane B is kept at 4° C., take membrane A out of the plate, processing with filter papers immersed in following solutions:10% SDS, 5 min, 0.5NNaOH/1.5MNaCl, 3 min, 1.5M NaCl/0.5M Tris.HCl, 3 min, 2×SSC/0.2M Tris.HCl, 10 min, dry at vacuum at 80° C., 2 hr, stored at 37C for the use.

[0042] (2) The preparation of gDNA probes

[0043] Sample 50-70ng of gDNA and make up water to ilml,boil at 100° C., 10 min, denature, use the following as primer marker reaction system: $\begin{matrix} {2\quad {mM}\quad {{dNTP}({dATP})}} & \quad & \quad & {3\quad {ul}} \\ {{primer}\quad {mixture}} & \quad & \quad & {2\quad {ul}} \\ {{klenow}\quad {enzyme}} & \quad & \quad & {1\quad {ul}} \\ {\alpha \text{-}{\,^{32}P}\text{-}{dATP}} & \quad & \quad & {3\quad {ul}} \end{matrix}$

[0044] mix up on vortex, incubate for 30 min at 37C,add 8ul stopmixture, filter through G-50 column to purify the probe, take one-tenth for liquid scintillation.

[0045] (3) Hybridization: colony dot matrix membrane is placed in 2×SSC and immersed for 10 min, remove carefully the debris on the surface of membrane, pre-hybridize at 65C in 5 ml hybridization liquid for 30 min at least. According to the value of liquid scintillation, based on 1.2×10⁶ cpm/ml hybridization liquid, sample probe liquid, boil at 100° C., 10 min, denature, add 5 ml fresh hybridization liquid to colony dot matrix membrane and allow for over 12 hr at 65C, and then wash the membrane under following conditions: 2×SSC/0.1% SDS, 10 min at room temperature, 2×SSC/0.1% SDS, 10 min at 65 C , 0.1×SSC/0.1% SDS, 10 min at 65° C. autoradiography at −70° C., strong or weak hybridization signal is considered to be single copy elementarily .

[0046] (4) Sequencing, southern blotting detection: pick clones without hybridization signal on the corresponding position of membrane, expand, extract plasmid for DNA sequencing, compared with database of GenBank, the copy without similarity to other sequence is considered to be single copy. Finally, isolate inserted DNA by enzyme digestion. Label the insert by α-³²P-dATP by random primer method and then hybridize with the EcoR1 digested gDNA on the nylon membrane, the clone showing one or two bands is considered to be single copy.

[0047] 1.3 Acquirement and identification of BM and short arms of group D, G chromosomes specific PAC clone

[0048] (1)Screen human PAC gDNA library to obtain positive clone

[0049] Using of α-³²P-dATP to label the single copy probe p8-7 of 260 bpby random primer method→purify the probe by G-50 column(middle size of particles)→keep for use at 4° C.→seven pieces of PAC membranes immersed in 2×SSC for→10 min pre-hybridize for 3 hr at 55° C.→denature probe for 10 min at 100° C.→add 50 ml hybridization solution purchased commercially according to dosage of 4.6×10⁵ cpm/ml,hybridize to PAC membrane for 1 hr at 65° C.→wash the membrane: 2×SSC, 10 min a time at ambient, wash with 2×SSC/0.1%SDS, 10 min at 65° C., twice placed onto the x-ray film, autoradiography for 12 hours→develop X-ray film count positive clone number as instruction goes.

[0050] (2) pick at random positive clones number from five different plates, purchase PAC clones.

[0051] 1.4 FISH of PAC DNA to metaphase cells the PAC confirms DNA was from group D,G chromosome, as shown in the FIG. 1. Experimental methods above referred to Molecular Cloning. Second Edition (Cold Spring Harbor Laboratory Press, 1989)

[0052] 2. Isolation of gene leading sequence DNA Main materials:β-agarase(Bio-Labs) Not I Agarase

[0053] {circle over (1)} Digest PAC 169 Plasmid by Not I enzyme;

[0054] {circle over (2)} Isolate Inserter DNA of 1 20 Kb by PFGE; Pulse electrophoresis conditions: electrode buffer solution:0.5×TBE, high strength Analytical Grade Agarase (Bio-Rad, Low Melting point Agarose LMP) 1%, Switch time:2 seconds→15 seconds→electrophoresis time:18 hr,voltage: 6V/cm, angle:120⁰ temperature: 14° C.

[0055] {circle over (3)} after clectrophoresis, stained with EB(0.2ug/ml),30 min,excise band of 120 kb.

[0056] {circle over (4)} the gel cut is treated with β-agarase, precipitated in alcohol .

EXAMPLE TWO

[0057] The preparation of gene vector provided by the present invention

[0058] 1. Construction of gene vector and transfer of gene of interest

[0059] 1.1 Construction of vector

[0060] 1.1.1 PAC DNA is digested by Nsi I and Stu I(blunt enzyme), 3.8 kb DNA fragment is recovered by general agarose gel electrophoresis, purification with electricity elution;

[0061] 1.1.2 Digest pGEM-TK vector DNA by Hind III, make it blunt by Klenow enzyme, to generate a blunt end;

[0062] 1.1.3 The linearized pGEM-TK /Hind III is further digested by Nsi I;

[0063] 1.1.4 The digested PAC DNA of 3.8 kb and pGEM-TK were ligated at 16° C. for 17 hr;

[0064] 1.1.5 Ligated product was transformed into JM109 competent bacteria, incubate in plate containing ampicillin for 18 hr at 37° C.;

[0065] 1.1.6 Pick monoclone at random, determine positive clones by Nsi I and Nhe I digestion after plasmid extraction, the recombinant plasmid was named pGEM-TK-3.8 Kb.

[0066] 1.1.7 Obtain Neo gene by digesting pCDN-GPR plasmid with Xba I and Nhe I

[0067] 1.1.8 Ligate Xba I and Nhe I digested Neo gene with Nhe I digested pGEM-TK-3.8 Kb to construct pNS2 gene vector;

[0068] 2.1 Transfer of TPA and FIX genes

[0069] 2.1.1 Clone TPA and FIX (CDS) into pcDNA3. I(−), respectively;

[0070] 2.1.2 Design the primers TPCF and TPCR to amplify TPA and FIX gene and expression elements(CMV promoter and BGH poly A signal),introduce enzyme Avr II restrictive sites into the two ends of primers, the sequences of primers go as following: TpcF: ATgCATCCTAggggAggTCgCTgAgTAgTg        AvrII TpcR: TgCATgCCTAggTACCCCCTAgAgCCCAg        AvrII

[0071] 2.1.3 Digests amplified TPA/FIX gene and expression components (CMV promoter and BGH poly A signal) by Avr II, and ligate it into pNS2 vector digested by NheI enzyme.

[0072] The procedures above, please see to “J.Sambrook et al. Molecular Cloning. Second Edition. Cold Spring Harbor Laboratory Press. 1989”

[0073]3.Extraction of gene vector DNA

[0074] 3.1 Materials

[0075] 3.1.1 QIAGE Plasmid Maxi Kit

[0076] 3.1.2 Culture Media: liquid LB Trypton   5 g Yeast extract  2.5 g NaCl  2.5 g Add ddH2O to  500 ml Autoclaved

[0077] 3.1.3 Ampicillin: 100 mg/ml(100×)

[0078] 3.2 Procedures

[0079] 1) Pick and inoculate positive clones into 3 ml LB(Amp⁺),incubate 1 hr at 37° C.

[0080] 2) put 100ul of primary culture above in 100 ml LB (Amp+), incubate 16 hr at 37° C.,250 rpm

[0081] 3) harvest bacteria by centrifugation at 6000g for 15 min at 4° C.

[0082] 4) add 100ml buffer P to resuspend bacterial pellet

[0083] 5) add 10 ml buffer P2, gently invert flask six times to mix up completely, stand for 5 min at ambient

[0084] 6) add 10 ml pre-cooled buffer P3, gently invert flask six times, place on ice for 20 min

[0085] 7) centrifuge at 20000g for 30 min at 4° C.

[0086] 8) transfer swiftly supernatant to 40 ml centrifugation tube, centrifuge at 20000g for 15 min at 4C

[0087] 9) 10 ml buffer QBT equilibrates QIGEN tip 500

[0088]10) transfer the supernatant to QIGEN tip 500,filter through the column

[0089] 11) wash the column with 2×30 ml buffer QC

[0090] 12) elute the column with 15 ml buffer QF, collect the elution liquid

[0091] 13) add isopropanol (0.7tinies volume) 10.7 ml to elution solution, thoroughly mix up

[0092] 14) centrifuge at 15000g for 30 min at 4° C.

[0093] 15) remove the supernatant, add 5 ml, 70% alcohol to DNA precipitate, centrifuge at 15000g, 4° C. for 10 min

[0094] 16) remove 70% alcohol,dry the DNA in the air for 10 min,add a certain amount of TE resolve DNA precipitate

EXAMPLE THREE

[0095] Introduce gene vector carrying TPA or FIX gene into host cells and expressed them in vitro

[0096] 1 Materials

[0097] 1.1 cell: HT1080 culture medium: high-sugar DMEM+10% FBS(HT1080)EMEM+10% FBS

[0098] 1.2 Electroporation apparatus: Bio-Rad company

[0099] 2.Methods:

[0100] 1) cells are inoculated in 75 cmn² canted-neck flask, culture and grow up to 70%-80% confluence

[0101] 2) Harvest the cells and wash twice with HeBs buffer solution, count the cell number

[0102] 3) Centrifugation at 1500 rpm, 4C for 10 min

[0103] 4) Resuspend with proper volume of HeBS, dilute the cell density to 10⁶-10⁷/ml

[0104] 5) Take 0.4 ml electroporation cuvette, add 0.8 ml cell suspension,10ul vector DNA

[0105] 6) Electroporate the cells at 260v, 550 uFf,lasting 11-13 ms

[0106] 7) The electroporated cells above are transferred into 75 cm² canted-neck flask, add 14 ml culture medium containing ampicillin/streptomycin, incubated in 5% CO₂,at 37° C.,24-48 hr.

[0107] 8) Add G418 to culture medium to a final concentration of 300ug/ml, screen, replace culture medium every 2-3 days and renew G418, HT1080 without gene transfer was used as control

[0108] 9) The control cells died after 7-10 days,count the survival clone cell number within transformed cells, and use maintenance volume of G418 of 150ug/ini

[0109] 10) Continue to screen transformed cells with GCV of 500ng/ml

[0110] 11) Most of cell clones died after 7-10days, add maintenance volume of GCV 250ug/ml, when the left survival cells grow up to 70%-80% confluence, detect the expression activity of transferred genes

[0111] 3.Results

[0112] TPA gene and FIX gene are introduced into HT1080cells, respectively by electroporation using human source gene vector, positive clones were obtained after positive and negative screening. Site-directed integration of two genes were confirmed by FISH (FIG. 3, 4). The results of activity determination detail as following table 1 and 2.

[0113] In negative control of HT1080 cells, TPA activity is 0 u /10 6 cells/24 hr, after the transfer TPA activity is 408 u /106 cells/24 hr,expression efficiency is 407 at day 95 after the transfer, the expression is very stable (Table 1). FIX activity is increased from less than 0.5ug/ml up to 2.5uVml, the expression content remain 2.6ug/ml,see Table 2.The expressing products of two genes have been testified by Western Blotting (Figure5,6),the expressed protein of TPA has been purified. TABLE 1 activity detection of positive cells transformed by TPA(ug/10⁶ cells/24 hr) Days after transformed T1 days after transformed T15 33 408 54 88 37 396 58 204 60 411 95 114 68 430 74 430 88 441 90 440.9 95 407

[0114] (T1, T15 are positive cell strains of tPA) TABLE 2 activity detection of FIX gene (ug/10⁶ cells/24 hr) Days after transformed clone F23 60 2.5 72 2.4 100 2.9 109 2.6

EXAMPLE FOUR

[0115] Safety case among human being

[0116] Prof. Xia Jiahui has been engaging in human and medical cytogenetics since 1973. He found and identified 732 of abnormal karotypes first reported in the world, which claimed by 470 clinical cytogeneticists who working with 189 laboratories around the China. Among these 732 karyotypes, 41 of which involve in short arms of D, G group chromosomes. No matter which chromosome is from chromosomes 1-22, the fragment translocated into short arms of group D, G chromosomes or the lengths of fragments from the same chromosome are different, the number of gene contained is from one to thousands, but the phenotype of the carrier is normal, which explains the genes translocated into short arms of D, G group chromosomes can express normally. So it is should be safe to use short arms of D, G group chromosomes as targeting sites for gene therapy.

[0117] 1. Karyotype:46,XX,t(1;12;22;15;11;8) (1qter→1p11::8p23→

[0118] 8pter; 12pter→12q 11::1p 11→1pter;22qter→22p11::12q11→

[0119]12qter;15pter→22p11→22pter; 11pter→11q21::15q15→

[0120] 15qter;8qter→8p23::11q21→11qter)

[0121] phenotype:female, 28 years old, normal phenotype carrier

[0122] material provider:Mfu subing, Cytogenetics laboratory, Department of gynecology and obstetrics, first affiliated hospital of Zhonrgshan Medical University,Guangzhou

[0123] 2. Karyotype: 46, XY,t(1; 13) (1pter→1q32::13p11→13pter; 13qter→

[0124] 13p11::1q32→1qter).

[0125] Phenotype:female, 24 years old, normal phenotype carrier

[0126] Material provider: Xiao Chen, Department of biology, Harbin Medical University,Harbin 150086

[0127]3.Karyotype: 46, XX, t(2; 15) (2pter→cen→15qter ;2qter→cen→15pter)

[0128] phenotype:female,26 years old, normal phenotype carrier

[0129] material provider:Guo Yuping, et al. Cytogenetics Department, Jiangxi provincial gynecology and obstetrics hospital, Nanchang 330006,Jiangxi province

[0130] 4.Karyotype: 46,XY,t(2;21) (2pter→cen→21pter; 2qter→cen→21 qter)

[0131] phenotype:male,32 years old, normal phenotype carrier

[0132] material provider: Kang Guoqing,et al. Department of genetics, the second affiliated

[0133] hospital of Shangxi Medical College, Taiyuan 030001

[0134]5. Karyotype: 46,XY,t(3;21) (2qter→cen→22pter; 3qter→cen→22qter)

[0135] phenotype:male,26 years old,normal phenotype carrier

[0136] material provider: Gao Yun.Department of toxicology,Bingzhou municipal Medical College, Bingzhou 256603, Shangdong province

[0137] 6. Karyotype: 46,XY,t(3;22) (3pter→cen→22pter; 3qter→cen→22qter)

[0138] phenotype :male,29 years old,normal phenotype carrier

[0139] material provider: Shi Huajin.Department of genetics,Jingzhou Women and Baby hospital,Jingzhou 121 000,Liaoning province

[0140] 7. Karyotype: 46,XX,t(4; 15)(4qter→4p13:: 15p 13→15pter; 15qter→

[0141]15p13::4p13→4pqter )

[0142] phenotype:female,28 years old, normal phenotype carrier

[0143] material provider: Zhou Ling,et al. Laboratory of genetics,the Wuhan Children hospital, Wuhan 430016,Hupei province

[0144] 8. Karyotype: 46,XYt(4;21)(4qter→4p15::21p11→21 pter;21qter

[0145] →21p11::4p 15→4pqter )

[0146] phenotype:female, 25 years old, normal phenotype carrier

[0147] material provider: Xu Jinfang, et al. Laboratory of genetics, the sixth people's hospital of Shanghai,Shanghai200000

[0148] 9. Karyotype: 46,XY,t(4; 14)(4qter→4q31:: 14p 11→14pter; 14qter→

[0149] 14p11::4q31→4qter)

[0150] phenotype: male, normal phenotype carrier

[0151] material provider: Zhou Mingjun,et al. Xuchang Municipal Central Hospital, Xuchang 161000,Henan province

[0152] 10. Karyotype: 46,XY,t(4;14)(4qter→4q35::14p11→14pter;14qter→

[0153] 14p11::4q35→4qter)

[0154] phenotype: male, 27 years old,normal phenotype carrier

[0155] material provider: Zhang Xiuquan,et al. Hushan Municipal Women and Nursling Hospital, Hushan 528000,Gtiangdonig province

[0156] 11. Karyotype: 46,XX, t(6; 22)(5qter→5q 13::22p11→22pter; 22qter→

[0157] 22p11::5q13→5qter)

[0158] phenotype: female, 32 years old, normal phenotype carrier

[0159] material provider: Zhao Jianping, Anyang Municipal Women and Nurslhg Hospital, Anyang455000,Henan province

[0160] 12. Karyotype: 46,XY, t(6;22) (6pter→cen6→22qter; 6qter→cen22→22pter)

[0161] phenotype:male,25 years old,normal phenotype carrier

[0162] material provider: Zhu Xinxia,et al. Laboratory of cytogenetics, Department of Gynecology and Obstetrics, Number 88 Hospital, Taian 271000,Shangdong province

[0163] 13. Karyotype: 46,XY, t(6; 22)(6qter→6p21::22p11.2→22pter; 22qter→

[0164] 22p11.2::6q21→6pter )

[0165] phenotype: male, 33 years old, normal phenotype carrier

[0166] material provider: Yang Qinglan, Department of Gynecology and Obstetrics, affiliated hospital of Bingzhou Medical College, Bingzhou 256603,Shangdong province

[0167] 14. Karyotype: 45,XX,t(7;21) (7qter→7p22::21p12→21qter)

[0168] phenotype:female,23 years old, normal phenotype carrier

[0169] material provider: Sun Qingji, et al. Laboratory of genetics, the Wuhan Children hospital, Wuhan 430016, Hubei province

[0170] 15. Karyotype: 46,XY/46XX,t(7;14)(7pter→7q 11::14p 11→

[0171] 14pter;14qter→14p 11::7q11→7qter )

[0172] phenotype: male,28 years old, normal phenotype carrier

[0173] material provider: Li Luyun, Xia Jiahui, et al. State Key Laboratory of Medical genetics(Hunan Medical University),Changsha 4 10078,Hunan province

[0174] 16. Karyotype: 46,XY,t(8; 14)(8pter→8p21:: 14p 12→14pter;14qter→

[0175] 14p12::8p13→8pter )

[0176] phenotype: male,27 years old, normal phenotype carrier

[0177] material provider: Shi I-Iuajin,et al. Department of genetics, Jingzhou Women and Baby hospital,Jingzhou 121000,Liaoning province

[0178] 17. Karyotype: 46,XY,t(9; 14)(9pter→cen→14pter; 9qter→cen→1 4qter)

[0179] phenotype: male,28 years old, normal phenotype carrier

[0180] material provider: Cheng Qiuyun,et al. Department of reproduction medicine, first affiliated hospital of Hengyang medical college, Hengyang 421001,Hunan province

[0181] 18. Karyotype: 46,XY,t(9;22) (9pter→9p13::22p12→22pter→22qter→

[0182] 22p12::9p13→9pter )mat

[0183] phenotype: female,31 years old, her mother, a young sister of her,a young brother of her and her son have the same phenotype as her, that is normal phenotype carrier material provider: Li Luyun,Xia Jiahui, et al. State Key Laboratory of Medical genetics(Hunan Medical University),Changsha 410078,Hunan province

[0184] 19. Karyotype:46,XX,t(9;14) (9pter→9q12::14p12→14pter; 14qter→

[0185]14p12::9q12→9qter).

[0186] Phenotype:female,32 years old, normal phenotype carrier

[0187] Material provider: Sun Yanyang,et al, Department of biology, Harbin Medical University, Harbin 150086

[0188] 20. Karyotype:46,XX,t(9; 15) (9pter→9q21::15p 12→15pter; 15qter→

[0189] 15p12::9q21→9qter)mat.

[0190] Phenotype:female,36 years old,normal phenotype carrier material provider: Zhu Guizhen,et al. Laboratory of cytogenetics, Department of Gynecology and Obstetrics, Number 88 Hospital, Taian 271000,Shangdong province

[0191] 21. Karyotype:46,XX,t(10;13) (10pter→10q24::13p 11→13pter; 13qter→

[0192] 13 p11::10q24→10qter)

[0193] Phenotype:female,28 years old,normal phenotype carrier material provider: Yan Dunqing. Department of Gynecology and Obstetrics,affiliated hospital of Qingdao Medical College,Qingdao266003,Shangdong province

[0194] 22. Karyotype: 46,XX,t(10; 13) (10pter→10q24::13p 12→13pter;13qter→

[0195] 13p12::10q24→10qter)

[0196] Phenotype:female,29 years old,normal phenotype carrier material provider: Zhang Yinru,et al. Department of neurology First affiliated hospital of Zhongshan Medical University,Guangzhou510080, Guangdong province

[0197] 23. Karyotype: 46,XX,t(11; 14) (11 pter→cen-14pter::11qter→cen→14qter) material provider:Wang Zhiyong,Department of genetics, Zhacheiig County people's hospital,Zhacheng County476200,Henan province

[0198] 24. Karyotype: 46,XX,t(11;21) (11pter→11p11::21p 11→21pter;21qter→

[0199] 21p11::11p→11pter)

[0200] Phenotype:female,26 years old,normal phenotype carrier material provider: Zheng Jun,et al. Department of genetics,Shanxi provincial women and nursling hospital, Xian7100O3 Shanxi province

[0201] 25. Karyotype); 46,XX,t(11;15) (11pter→11q13::15p12→15pter;15qter→

[0202]15p12::11q13→11qter)

[0203] Phenotype: male,23 years old, normal phenotype carrier

[0204] material provider: Yang Ruifang,et al. Medical center of Obstetrics,affiliated hospital of Shandong Medical University,Jinan250012, Shandong province

[0205] 26. Karyotype:46,XX,t(12; 14) (12pter→cen→14pter::12qter→cen→14qter)

[0206] Phenotype:female,28 years old, normal phenotype carrier material provider: Han Weitian,et al. Department of eugenics,Liaoning provincial institue of family planing, Shenyang 110031,Liaoning province 27. Karyotype:46,XX,t(13;16) (13qter→13p 11::16p11.2→16pter; 16qter→

[0207] 16p11.2::13p11→13pter

[0208] Phenotype:female,27 years old, normal phenotype carrier

[0209] material provider: An Songlan. Department of genetics, Dalian municipal gynecology and obstetrics, Dalian 110078,Liaoning province

[0210] 28. Karyotype: 46,XY/46,XX,t(13;13) (13qter→13p12::13p12→13qter)

[0211] Phenotype: male, 39 years old, normal phenotype carrier

[0212] material provider: Li Luyun, Xia Jiahui, et al. State Key Laboratory of Medical genetics(Hunan Medical University),Changsha 410078,Hunan province

[0213] 29. Karyotype:46,XY,t(14; 18) (14pter→cen→18pter; 14qter→cen→18qter)

[0214] Phenotype: male,30 years old, normal phenotype carrier material provider: Wang Sugui, et al. Beijing Institute of family planing technology guidance, Beijing 100006

[0215] 30. Karyotype:46,XX,t(14; 15) (14pter→14q13::15p 13→15pter;15qter→

[0216]15p13::14q13→14qter)

[0217] Phenotype:female,28 years old, normal phenotype carrier material provider: Li Luyun,Xia Jiahui, et al. State Key Laboratory of Medical genetics(Hunan Medical University),Changsha 410078,Hunan province

[0218] 31. Karyotype:46,XX,t(15qter→cen→22qter)

[0219] Phenotype:female,27 years old, normal phenotype carrier material provider: Li Luyun,Xia Jiahui, et al. State Key Laboratory of Medical genetics(Hunan Medical University),Changsha 410078,Hunan province

[0220] 32. Karyotype:46,XY,t(15; 18) (15pter→cen→18pter; 15qter→cen→18qter)

[0221] Phenotype: male,30 years old, normal phenotype carrier

[0222] material provider: Ren Guoqing,et al. Beijing Institute of family planing technology guidance, Beijing 100006

[0223] 33. Karyotype:46,XX,t(15 ;20) (15pter→cen→2pter,; 15qter→cen→2qter)

[0224] Phenotype:female,26 years old, normal phenotype carrier material provider: Wang Xin, et al. Laboratory of genetics, department of obstetrics, the second affiliated hospital, Hunan Medical University,Changsha 410011,Hunan province

[0225] 34. Karyotype:46,XX,t(15;22) (15pter→15q11: :22p13→22pter;22qter→

[0226]22p13::15q11→15qter)

[0227] Phenotype:female,27 years old, normal phenotype carrier

[0228] material provider: Hu Shengdi, Department of genetics, Hainan provincial people's hospital, Haikou570011,Hainan province

[0229] 35. Karyotype:46,XX,t(15;22) (15pter→15q22: :22p11→22pter;22qter→

[0230]22p11::15q22→qter)

[0231] Phenotype:female,29 years old, normal phenotype carrier

[0232] material provider:Li Murou, Department of genetics, Xinjiang Medical College, Urumchi 830054

[0233] 36. Karyotype:46,XY,t(16;21) (16pter→16q11::21p 11→21pter;21qter→

[0234]22p11::16q12→16qter)

[0235] Phenotype: male,29 years old, normal phenotype carrier

[0236] material provider: Zhang Huifang,et al, Institute of family planing technology of Guangdong,Guangzhou510080,Guangdong province

[0237] 37. Karyotype:46,XX,t(18;21) (18pter→cen→21 pter; 18qter→cen→21 qter)

[0238] Phenotype:female, normal phenotype carrier

[0239] material provider: Shi Huajin, et al. Laboratory of genetics, Jingzhou women and nursling hospital, Jingzhou 121000,Liaoning province

[0240] 38. Karyotype:46,XX, t(1 8;21) (18pter→18q11::21p12→21 pter;21qter→

[0241]22p12::18q11→18qter)

[0242] Phenotype: female,26 years old, normal phenotype carrier material provider:Li Xiulin,et al,laboratory of genetics, department of pediatrics,first affiliated hospital of Chinese medical university,Shenyang 10011 1,Liaoning province

[0243] 39. Karyotype:45,X,dic(Y;13)(Ypter→Yp 1200::13p 11→cen→13qter)

[0244] Phenotype: male,4 years old, normal phenotype carrier

[0245] material provider: Xia Jiahui, et al. State Key Laboratory of Medical genetics(llunan Medical University),Changsha 410078,Hunan province

[0246] 40. Karyotype:46,XY,t(Y;15)(15qter→15p12::Yq12→Ypter) pat.

[0247] Phenotype: male,4 years old, normal phenotype carrier

[0248] material provider: Xia Jiahui, et al. State Key Laboratory of Medical genetics(Hunan Medical University),Changsha 410078,Hunan province

[0249] Abnormal chromosomes carriers above showed no any abnormal syndrome, which explains that not only nucleolus tissue can receipt foreign genes but also allow foreign genes to express normally. 

1. The leading sequence of human source gene vector comes from short arms of human D, G group Chromosomes where there is no pivot physiological function-related gene sequence, or a DNA sequence with 50% or over 50% identity to the leading sequence.
 2. The leading sequence of human source gene vector according claim 1 further characterized by the said sequence selected from the DNA sequence in SEQ No. 1 or a human DNA sequence with 50% or over 50% identity to the leading sequence.
 3. The leading sequence of human source gene vector according claim 2 further characterized by the said sequence at positions from 75590 to 79448 in SEQ No. 1 or a DNA sequence with 50% or over 50% identity to the 3.8 Kb of the said DNA sequence.
 4. The gene vector the leading sequence of which is the sequence described in the claim 1 or claim
 2. 5. The gene vector according to claim 4 wherein that leading sequence is at the positions from 75590 to 79448 in sequence list 1 or 50% or a DNA sequence with 50% or over 50% identity to the 3.8 Kb of DNA sequence.
 6. The gene vector according to claim 5 which contains positive and negative screening genes.
 7. The gene vector according to claim 6 wherein DNA sequence of the vector is that in SEQ No. 2, using TK as negative screening gene, insertion of positive screening gene Neo into position 1500 of GLS, the GLS is divided into two arms of 1.5 kb and 2.3 kb to construct the gene vector, cloning site of the gene of interest is
 5910. 8. The strategies of gene expression include following procedures: (1) Using DNA sequence without pivot physiological function-related gene in short arms of human D, G group chromosomes or a sequence with 50% or over 50% identity to the selected DNA sequence above for gene vector construction. (2) Clone the gene of interest-into the gene vector; (3) Transduce the gene of interest into the target region of D, G group chromosomes of human cells; (4) The gene of interest expresses in vivo or in vitro cells.
 9. The strategies of gene expression according to claim 8 wherein the vector is that the leading sequence of the gene of interest is selected from the DNA sequence of SEQ No. 1 or the DNA sequence which is 50% or over 50% homologous.
 10. The strategies of gene expression according to claim 9 wherein the leading sequence of the gene of interest is at the positions from 75590 to 79448 in the SEQ No. 1 or to which the DNA sequence is 50% or over 50% homologous.
 11. The strategies of gene expression according to claim 10 wherein the gene vector contains the positive and negative screening genes.
 12. The strategies of gene expression according to claim 11 wherein the DNA sequence of the gene vector is that in SEQ No. 2, the leading sequence is at the positions from 75590 to 79448 in SEQ No. 1, using TK as negative screening gene, insertion of positive screening gene Neo into the position 1500 of GLS, the GLS is divided into tow arms 1.5 kb and 2.3 kb, finish the construction of the gene vector, the site of the gene of interest is
 5910. 